br Autophagy assay br Autophagic vacuoles labeled with monod
2.7. Autophagy assay
Autophagic vacuoles labeled with monodansylcadaverine (MDC, Sigma Brazil) according to what previously described with minor mod-ifications . Cells (MDA-MB-231 and MCF10A) were seeded in 24-well plates containing, sterile glasses coverslips and allowed to attach overnight. Afterwards, Sorafenib were treated with BthTX-II (50 μg/ml) for 24 h and compared with untreated cells. After treatment, the cells were incubated with 0.05 mM MDC in PBS at 37 °C for 30 min. After re-moving the MDC, the cells fixed in 4% paraformaldehyde for 15 min at r> 37 °C. The samples were observed using fluorescence microscopy (Zeiss LSM510, Germany). Using the support of Image J software (Bethesda, MD, USA), four randomly selected microscopic fields at 10 X magnifica-tion were used to quantify the fluorescence intensity, which represents the relative levels of cellular autophagy. The average fluorescence values were calculate from triplicates.
2.8. Apoptosis analysis
MDA-MB-231 cells (2 × 105/well) were seeded in 12-well tissue cul-ture plates, and incubated for 18 h at 37 °C and 5% CO2. Next, cells were treated for 24 h. Apoptosis was measured after 24 h of treatment with BthTX-II (10 and 50 μg/ml) by using the Annexin V PE/Iodide propidium apoptosis kit (BD Pharmingen™). Untreated cells were used as control. Cells were analyzed by flow cytometry (BD Accuri C6, USA) and graphs were obtained by using the software FlowJo.10 (Treestar Inc., San Car-los, CA).
2.9. Cell cycle analysis
MDA-MB-231 cells (2 × 105 cells/ml) seeded in 12-well plates and were G0 synchronized by serum starvation for 24 h. Next, cells were treated with two different concentrations of BthTX-II (10 and 50 μg/ ml) in medium supplemented with 10% FBS. After 24 h of treatment, the cells were collected by using a scraper, fixed in 80% ethanol, washed with PBS and treated with RNAse (Sigma-Aldrich, Brazil). Afterwards, cells were stained with propidium iodide (PI) for 15 min (Sigma-Al-drich, Brazil) and analyzed by flow cytometry (BD Accuri C6, USA).
2.10. Gene expression analysis for real-time PCR
MDA-MB-231 cells incubated with BthTX-II (50 μg/ml) and with complete medium (control group) for 24 h, at 37 °C and 5% CO2. The total RNA was extracted using Trizol reagent (Invitrogen, Brazil). The extracted RNA quantified, and reverse transcribed into cDNA using a Promega GoScript reverse transcription kit, according to the manufac-turer's instructions. Real-time PCR analysis were carried out in triplicate using SYBR Green PCR master Mix (LGC, Biotechnology, Brazil) as a fluo-rescent dye in a thermal Cycler (ABI 7300-Applied Biosystem, USA). The primers were purchased from Human Cancer Pathway Primer (GO GENONE-HCAN-1®, Brazil). The results were normalized using GAPDH as a housekeeping gene, subsequently calibrated with the normalized data of the control sample to determine the relative quantification = 2(−Delta Delta C(T)) method .
2.11. Cell adhesion inhibition assay
To evaluate the interaction between BthTX-II and extracellular ma-trix components (ECM), collagen IV (10 μg/ml in 0.1 M of acetic acid), fibronectin (10 μg/ml in PBS), and Matrigel (1 mg/ml in PBS) were coated on 96-well plates overnight at 4 °C. Subsequently, MDA-MB-231 cells previously incubated for 30 min at 37 °C with different concen-trations of BthTX-II (2.5, 10, 25, and 50 μg/ml) and untreated cells were added to a 96-well plate for 3 h at 37 °C in 5% CO2. The adhering cells were quantified by MTT assay and intensity was measured at 570 nm using a microplate reader (Multiskan GO Thermo Scientific, USA).
In order to analyze MDA-MB-231 cells migration, the wound-healing assay was used according to what previously described  with minor modifications. Briefly, 1 × 105 cells/well seeded in 24-well tissue culture plates and incubated overnight. The medium was discarded and then a wound was created by scratching the cell mono-layer with a 10 μL pipette. Subsequently, cells were treated with BthTX-II (10 and 50 μg/ml) or medium only (control) for 24 h. Cell
migration was analyzed by comparing cells photographed at 10× mag-nification (Nikon Eclipse TS100) immediately after scratching (t = 0) and upon 24 h of treatment (t = 24 h). The closure of the wound was quantified by the software Image J (NIH, MD, USA) and the percentage value was obtained by applying the formula (F1) 
2.13. Transwell migration inhibitions assay
The migration assay was performed using 300 μl of cell suspension of MDA-MB-231 (7 × 104 cells/well) pre-incubated in serum-free medium containing BthTX-II (10 and 50 μg/ml) for 30 min at 37 °C. Cells were seeded in the upper chamber of each insert of the transwell polycarbon-ate membrane with 8.0-μm; (Greiner Bio-one, Switzerland). The lower chamber was filled with medium containing 10% FBS as chemoattractant. After culturing for 24 h, the non-migrating cells were removed with a cotton-tipped applicator using the upper inserts, then air-dried, fixed and stained with a panotic kit. Six fields of view were randomly selected for cell counting under a microscope at 10× magnification. Cell migration was described as the relative numbers of transmitted cells.